Identification of miR-27b as a novel signature from the mRNA profiles of adipose-derived mesenchymal stem cells involved in the tolerogenic response

Abstract

Adipose-derived mesenchymal stem cells (adipose-derived MSCs, ASCs) possess the ability to differentiate into multiple tissue types and have immune-modulatory properties similar to those of MSCs from other origins. However, the regulation of the MSC-elicited immune-modulatory activity by specific microRNA (miRNA) mechanisms remains unexplored. Gene expression profiling with knowledge-based functional enrichment analysis is an appropriate approach for unraveling these mechanisms. This tool can be used to examine the transcripts and miRNA regulators that differentiate the rat tolerogenic orthotopic liver transplantation (OLT; DA liver into PVG) and rejection OLT (DA liver into LEW) models. In both models, the rejection reaction was observed on postoperative day 7∼14 (rejection phase) but was overcome only by the PVG recipients. Thus, the global gene expression patterns of ASCs from spontaneously tolerant (PVG) and acute rejecting (LEW) rats in response to LPS activation were compared. In this study, we performed miRNA enrichment analysis based on the analysis of pathway, gene ontology (GO) terms and transcription factor binding site (TFBS) motif annotations. We found that the top candidate, miR-27, was specifically enriched and had the highest predicted frequency. We also identified a greater than 3-fold increase of miR-27b expression in the ASCs of tolerant recipients (DA to PVG) compared to those of rejecting recipients (DA to LEW) during the rejection phase in the rat OLT model. Furthermore, our data showed that miR-27b knockdown has a positive influence on the allosuppressive activity that inhibits T-cell proliferation. We found that miR-27 knockdown significantly induced the expression of CXCL12 in cultured ASCs and the expression of CXCL12 was responsible for the miR-27b antagomir-mediated inhibition of T-cell proliferation. These results, which through a series of comprehensive miRNA enrichment analyses, might be relevant for stem cell-based therapeutic applications in immunosuppressive function using ASCs.

Figure 1. Overlapping differentially expressed genes identified by comparisons between the ASCs of the LEW and PVG groups and the top interacting network among the shared genes identified by the 3 comparisons.

(A) A Venn diagram detailing the number of differentially expressed genes that were shared and distinct between three comparisons (List 1: comparing expression in the LEW-LPS group at 24 h and the PVG-LPS group at 24 h. Genes with greater than a 1.5-fold change were identified with the Mann-Whitney unpaired test(p<0.05). List 2: comparing the LEW and PVG groups with or without LPS stimulation at 24 h. Genes with greater than a 1.5-fold change at least 1 condition were considered. List 3: results of the 2-way ANOVA with Benjamini-Hochberg multiple testing correction (p<0.05). The number of transcripts within each subset is highlighted in the yellow boxes adjacent to the Venn diagram. The number of genes shared by at least 2 of the 3 gene lists is indicated by the purple color. The 358 total genes were subjected to further network analysis. (B) The functional interaction networks among the 358 genes were analyzed using the significant pathway analysis tool in Genespring GX. The top networks of significant “direct interactions” and their associated p-values are shown. (C) Complete hierarchical linkage analysis was applied to the 16 genes identified in the top network. Each column represents an individual pooled sample, and each row represents a specific gene. Red indicates high relative expression, and blue indicates low relative expression. The upper panel depicts 9 genes that are responsible for low expression levels in the ASCs of the LEW rats (cluster 1). The lower panel depicts 7 genes that are differentially expressed after LPS treatment. The relative gene expression levels of the 16 genes are also presented (D).

Figure 2. The less-stringent procedure of the knowledge-based enrichment analysis for detecting differentially expressed genes in the ASCs from the LEW and PVG rats.

A Venn diagram showing the number of gene transcripts that were shared and distinct among the differentially expressed genes identified using the less-stringent statistical criteria. The number of annotated gene transcripts resulting from each step is given in purple. The application of the model-free categorical analysis for GO enrichment was performed using the online GOToolBox software.

Figure 3. miR-27b expression in response to LPS treatment in the ASCs isolated from the LEW and PVG recipients after orthotopic liver transplantation (OLT).

Isolated ASCs from the LEW and PVG recipients after OLT were incubated with 200 ng/ml LPS for 6 or 24 h. Total RNA was purified from the respective cell pellets, and the expression of miR-27b was analyzed by quantitative RT-PCR. The miRNA expression was normalized with U6. All results are expressed as the mean ± SD from three independent experiments. *p<0.05 compared with untreated ASCs.

Figure 4. miR-27b regulates ASC-mediated suppression of CFSE-labeled allogeneic CD4⁺ T cell proliferation.

(A) ASCs from Lewis rats were transfected with α-miR-27b or α-miRNC (25 µg/ml) for 24 h. Cells were harvested and the relative expression levels of CXCL12 mRNA were measured. Western blot analysis was also performed for CXCL12 and actin (as internal control). (B) A total of 2.5×10⁻⁴ purified splenic T cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE). The cells were then cultured with Con A (1 µg/ml) (as 100% proliferation) and Con A + with different ratios of ASCs (1∶10, 1∶5, 1∶1, ASCs:T cells) with α-miR-27b or α-miRNC transfection. The cells were then stained with the anti-CD4 antibody, and proliferation was analyzed by FACS. A decrease in CFSE staining is an indication of proliferation. We quantified the percentage of CD4⁺ cells that have low levels of CFSE labeling. (C) Knocking out CXCL12 by siRNA transfection significantly impaired the suppressive actions of ASCs on T cell proliferation. Data are expressed as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 compared with untreated ASCs.